1. Updates on Prion Protein Purification Donovan Duggan Bow Suriyamongkol Supervisor: Dr. David Wishart Prion Group Meeting 2 February, 2007

2. Donor

3. Purification continues

4. Optimisation of purification Steps to remove contaminating upper and lower bands Gradient Elution Possible double his tag, combined with a gradient elution

5. Post purification protocols 1) Determine protein concentration 2) Determine optimal buffer for highly concentrated samples 3) Determine protein structure during each step of post purification protocols

6. 1) Determine protein concentration All quantification methods are rough approximations. Absorbance at 280 Hartree-Lowry and modified Lowry Protein assays. Bicinchoninic Acid (BCA) Bradford Kjeldahl Very rough approximations with visualization in gels or on filter papers.

7. 2) Determination of optimal buffers Heavy empirical screen. Temperature pH Ionic strength Detergents Glycerol content Small stabilizing molecules

8. 3) Determine protein configuration before and after any condition change. Circular Dichroism Microscopy

9. Wild Type (3 mg/mL)

10. F198S (4 mg/mL and 2 mg/mL)

11. D178N (4 mg/mL)

12. E200K (2 mg/mL)

13. Summary of Solubility of The Proteins Wild type (2 mg/mL), pI 8.86  almost all the buffers tested, including ddH2O F198S (2 mg/mL), pI 8.86  20 mM Tris, 100 mM NaCl, pH 8.0 D178N (4 mg/mL), 9.08  10 to 50 mM NaOAc, pH 4.0-5.0 E200K (2 mg/mL), 9.23  10 to 50 mM NaOAc, pH 4.0-5.5

14. Donovan’s results for P102L mutant Sodium acetate buffer (up to 3 days at 37C) - 10 to 100 mM, pH 4.0 - 25 to 100 mM, pH 4.5 - 50 to 100 mM, pH 5.0 - 10 to 50 mM, pH 5.5 P102L ( ? mg/mL) is stable in: Potassium phosphate buffer (up to 1 day at 37C) - 10 to 100 mM, pH 8.0

15. SDS-PAGE Gels of Purified Proteins Wild type 15 20 kDa Total amount of 45 milligrams per 1L of culture F198S 15 20 kDa Total amount of 35 milligrams per 1L of culture

16. Protein Quantification

17. Acknowledgements Dr. David Wishart Valentyna Semenchenko Thank you.